The aminoacyl-tRNA synthetases are responsible for pairing amino acids and tRNAs during translation and are crucial in defining the genetic code. Aminoacyl-tRNA synthetases normally belong to one of two unrelated structural classes (I and II), the only known exception to this rule being the lysyl-tRNA synthetases. Consistent with their exceptional structural and functional diversity, the lysyl-tRNA synthetases have been implicated in a wide variety of cellular roles separate from aminoacyl-tRNA synthesis ranging from antibiotic resistance to translational control. Many of these roles depend on the ability of lysyl-tRNA synthetases to form functional interactions with other proteins, providing a model system to study non-canonical roles of the aminoacyl-tRNA synthetases. The objectives of this proposal are to: i) Determine how interactions between class I and II lysyl-tRNA synthetases lead to the aminoacylation of non-canonical tRNAs. ii) Investigate the role of lysyl-tRNA synthetases in establishing antibiotic resistance. iii) Probe the function of lysyl-tRNA synthetase in determining translational efficiency as a component of a multi-aminoacyl-tRNA synthetase complex. The results of these experiments will reveal roles for lysyl-tRNA synthetases in cellular processes separate from translation, and show how lysyl-tRNA synthetases provide new and altered functions by forming complexes with other proteins. Project Narrative Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus are common agents of foodborne disease with potentially fatal sequelae. The class 1 and class 2 lysyl-tRNA synthetases contribute to the virulence of these bacterial pathogens by modulating antibiotic resistance through mechanisms which to date have only been minimally defined. The proposed studies will define the roles of class 1 and class 2 lysyl-tRNA synthetases in establishing antibiotic resistance, and help determine if the corresponding pathways can be used as targets for anti-infective agents. The study will also provide further insights into the degree of functional diversity within the aaRS family in general, an emerging group of anti-microbial drug targets.